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Flow Cytometric Detection of Lymphoma MRD
Maryalice Stetler-Stevenson, M.D., Ph.D. Director Flow Cytometry Laboratory,
Laboratory of Pathology, NCI, NIH
DEPARTMENT OF HEALTH & HUMAN SERVICES
National Institutes of Health Bethesda, Maryland 20892
Public Health Service
Basis for NCI Approach to Mature Lymphoma/Leukemia MRD
Mature B-cell and T-cell lymphomas/ leukemias/ have aberrant antigen expression
The aberrant antigen expression allows one to detect MRD in the presence of polyclonal B and T-cells
Currently greater sensitivity is achieved with B-cell than with T-cell lymphoma.
Detection of Mature Lymphoma/Leukemia MRD with Known Specific IP
CD5+ B-cell neoplasia: CLL, mantle Cell lymphoma
CD11c+ B-cell neoplasia: Hairy cell leukemia, Hairy cell leukemia variant, some splenic marginal zone lymphoma
CD10+ B-cell neoplasia: Follicular lymphoma, Burkitt lymphoma
Mycosis Fungoides
SF08 00901.006 L FSC/SSC
CD19 PerCP Cy5.5
CD
5 A
PC
100 101 102 103 104100
101
102
103
104
20.89%53.23%
0.91%24.97%
SF08 00901.006 VIABLE by FSC/SSC
CD19 PerCP Cy5.5
SSC
-Hei
ght
100 101 102 103 1040
256
512
768
1024
SF08 00901.006 B CELLS CD19PERCP
CD19 PerCP Cy5.5C
D5
APC
100 101 102 103 104100
101
102
103
104
Gate 66
SF08 00901.006 CD5+CD19+
KAPPA MONO FITC
LAM
BD
A M
ON
O P
E
100 101 102 103 104100
101
102
103
104
Detection of CD5 Positive MRD Among Polyclonal B-Cells
Patient with history of mantle cell lymphoma
Detection of CLL MRD Among Polyclonal B-Cells
CD19 PC7
CD
5 A
PC
10 2 10 3 10 4 10 5
102
103
104
105
86.60%0.00%
13.40%0.00%
Kappa-m FITC
Lam
bda-
m P
E
10 2 10 3 10 4 10 5
102
103
104
105
No MRD detected 0.006% CLL MRD
SF13 1132 pb MRD 6_01_S-1.fcs
CD3 PerCP
CD
19 P
E
10 2 10 3 10 4 10 5
102
103
104
105
75.19%16.64%
0.02%8.15%SF13 1132 pb MRD 6_06_B-3.fcs
CD19 PC7C
D38
v45
010 2 10 3 10 4 10 5
102
103
104
105
SF13 1132 pb MRD 6_06_B-3.fcs
Kappa-m FITC
Lam
bda-
m P
E
10 2 10 3 10 4 10 5
102
103
104
105
SF13 1132 pb MRD 6_06_B-3.fcs
CD20 PerCP
CD
5 A
PC
10 2 10 3 10 4 10 5
102
103
104
105
SF13 1132 pb MRD 6_06_B-3.fcs
Kappa-m FITC
Lam
bda-
m P
E
1 0 2 10 3 10 4 1 0 5
10 2
10 3
10 4
10 5
FSC-A
SSC
-A
0 52429 104858 157286 209715 262144
0
52429
104858
157286
209715
262144
P1
CD3 v500
CD19
PC7
10 2 10 3 10 4 10 5
102
103
104
105
32.56%54.90%
0.10%12.44%
CD81 FITC
CD
43 A
PC
10 2 10 3 10 4 10 5
102
103
104
105
P2
SSC-A
CD
43 A
PC
0 52429 104858 157286 209715 262144
102
103
104
105
20.18%75.79%
3.96%
0.07%
CD20 AH7
CD
22 P
erC
P C
y55
10 2 10 3 10 4 10 5
10 2
10 3
10 4
10 5 P4
CD79b PE
CD
5 v4
50
10 2 10 3 1 0 4 10 5
102
103
104
105
P3
Q2
Q4Q3
Q1
Q4-1
Q1-1
Q3-1
Q2-1
Gate 2:Cells in quadrant Q1-1 and within Q1 and Q3
Gate 1:Cells in quadrants Q1 and Q3
Gate 3:Cells in P1 and within Q1-1 , Q1 and Q3
Combined Analysis Gate: Cells in Q1, Q3, Q1-1, P1, P2, P3 and P40.007% of cells in Combined Analysis Gate
Detection of CLL MRD Among Polyclonal B-Cells- ERIC Method
Detection of CD11c Positive MRD Among Polyclonal B-Cells
CD19 PC7
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
8c B CD19 PC7 SSC
CD103 FITC
CD
123
PerC
P C
y55
102
103
104
105
102
103
104
105
CD25 PE
CD
11c
V450
102 103 104 105
102
103
104
105
CD20 AH7
CD
11c
V450
102 103 104 105
102
103
104
105
Kappa-m FITC
CD
11c
V450
102 103 104 105
102
103
104
105
Lambda-m PE
CD
11c
V450
102 103 10 4 105
102
103
104
105
CD103 FITC
CD
11c
V450
102 103 104 105
102
103
104
105
Kappa-m FITC
Lam
bda-
m P
E
102 103 104 105
102
103
104
105
Detection of CD10 Positive MRD Among Polyclonal B-Cells
SF12 653 pb_07_B-4.fcs
Kappa-m FITC
Lam
bda-
m P
E
102
103
104
105
102
103
104
105
SF12 653 pb_07_B-4.fcs
CD19 PC7
SSC-
A
102 103 104 1050
65536
131072
196608
262144
8c B CD19 PC7 SSC
SF12 653 pb_07_B-4.fcs
Kappa-m FITC
CD
10 A
PC
102
103
104
105
102
103
104
105
SF12 653 pb_07_B-4.fcs
Lambda-m PE
CD
10 A
PC
102
103
104
105
102
103
104
105
SF12 653 pb_07_B-4.fcs
Kappa-m FITC
Lam
bda-
m P
E
102
103
104
105
102
103
104
105
SF12 653 pb_01_S-1.fcs
CD3 PerCP
CD
19 P
E
102
103
104
105
102
103
104
105
SF12 653 pb_01_S-1.fcs VIABLE by FSC/SSC
FSC-A
SSC
-A
0 65536 131072 196608 2621440
65536
131072
196608
262144
8c L FSC SSC
SF12 653 pb_07_B-4.fcs
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105
SF12 653 pb_07_B-4.fcs
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105
Mycosis fungoides typically expresses dimmer CD3 than normal T-cells, and is CD4 positive but is negative for CD7 and CD26 CD26 is positive in the vast majority of CD4 positive T-cells
ATL- Adult T-cell Leukemia/Lymphoma associated with HTLV-1 has the same IP but is also CD25 bright
Detection of Mycosis Fungoides MRD:
CD3 APC
CD4
PerC
P
100 101 102 103 104100
101
102
103
104
CD3 APC
CD26
FIT
C
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD7
FITC
100 101 102 103 104100
101
102
103
104
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsSpecific populations normally
present in low numbersRestricted Populations
Detection of T Cell Neoplasia:Absence of Normal Antigen
75% of mature T cell neoplasms missing a normal antigen CD7 is most frequent missing antigen
Commonly absent in subset of normal T-cells
CD5 or CD2 second most commonCD5 absent in subset of gamma delta T
cells CD3 lowest if include cytoplasmic CD3 CD4 and CD8 negative low in mature
tumors
Detection of T Cell Neoplasia:Absence of Normal Antigen
CD3 APC
CD
4 FI
TC
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
8 PE
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD
2 PE
1:1
S5.
2
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD
5 A
PC
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD
7 FI
TC
100 101 102 103 104100
101
102
103
104
69 yo male with enlarged left cervical lymph node. FNA of lymph node submitted for flow cytometry
Final Diagnosis: PTCL NOS. Malignant cells are CD2+, CD3 dim, CD5-, CD7-, CD4- and CD8 dim
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsSpecific populations normally
present in low numbersRestricted Populations
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
Detection of T Cell Neoplasia: Presence of Abnormal Antigens
CD3 APC
CD19
Per
CP100 101 102 103 104
100
101
102
103
104CD19 ExpressionFNA: CD19+ PTCL
CD10 Expression
CD5 FITC
CD
10 A
PC
100
101
102
103
10410
0
101
102
103
104
CD3 PerCP
CD
5 A
PC
100
101
102
103
10410
0
101
102
103
104
CD3 PerCP
CD
2 PE
100
101
102
103
10410
0
101
102
103
104
FNA: AILT:CD3-, CD5+, CD7-, CD2+, CD4+, CD10+
Rizzo, Stetler-Stevenson, Wilson, Yuan, Clinical Cytometry, 2009; 76B:142-149Yuan et al, Human Pathology, 2005;36:784-791
A 65 yo woman- multiple erythematous 2 cm to 7 cm tumors on the upper extremities for about one year. No lymphadenopathy or organomegaly. FNA of skin lesion sent to flow cytometry.
CD45 PerCP
CD
4 FI
TC
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
4 FI
TC
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
8 PE
100 101 102 103 104100
101
102
103
104
CD4 PerCP
CD
30 F
ITC
100 101 102 103 104100
101
102
103
104
CD5 APC
CD
2 PE
BD
S5.
2
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD
7 FI
TC
100 101 102 103 104100
101
102
103
104
CD30 FITC
CD
2 PE
S5.
2
100 101 102 103 104100
101
102
103
104
CD30+, CD2+, dim CD4+, dim CD45+ and CD3-, CD5-, CD7- and CD8- Primary Cutaneous CD30+ T cell LPD (ALCL). Juco, J. Holden, K.P. Mann, L.G. Kelly, S. Li, AJCP, 2003:119:205-212
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsSpecific populations normally
present in low numbersRestricted Populations
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
Detection of T Cell Neoplasia:Abnormal Antigen Intensity
CD5 APC
CD
7 FI
TC
100 101 102 103 104100
101
102
103
104
CD3 PerCPC
D2
PE 1
:1 S
5.2
100 101 102 103 104100
101
102
103
104
T Cell Lymphoma: CD3-, CD7-, CD5 bright, CD2 bright
Detection of B Cell Neoplasia:Abnormal Antigen Intensity
Kappa
Lambda
B Cell Gate
CD19
CD3
CD19
CD3
Lambda
Kappa
Bright CD19 Gate
SF08 00901.007
CD20 PerCP
CD
5 A
PC
100 101 102 103 104100
101
102
103
104
SF08 00901.007
CD22 PE
SSC
-Hei
ght
100 101 102 103 1040
256
512
768
1024
SF08 00901.008
CD20 PerCP
Lam
bda
KA
LL F
itc
100 101 102 103 104100
101
102
103
1040.00% 12.00%
0.00% 88.00%
SF08 00901.007
CD20 PerCP
Kap
pa K
ALL
Fitc
100 101 102 103 104100
101
102
103
104
14.57%0.00%
85.43%0.00%
Detection of B Cell Neoplasia:Abnormal Antigen Intensity
Dim CD20 and CD5+
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsSpecific populations normally
present in low numbersRestricted Populations
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
Abnormally Large T Cells:
FSC-Height
SSC
-Hei
ght
0 256 512 768 10240
256
512
768
1024
CD3 APC
CD
4 FI
TC
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
8 PE
100 101 102 103 104100
101
102
103
104
Large Cells (High FSC) are CD3 dim+, CD4+, CD8dim to -
Abnormally Large B Cells:
Kappa
Lambda
Small polyclonal B-cells
Lambda
Kappa
Small T-cells
Lambda
Kappa
CD20-PE
FSC-Size
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsSpecific populations normally
present in low numbersRestricted Populations
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
21yo BM with hepatosplenomegaly: Gamma delta T cell lymphoma
TCRab +, CD3+, CD57-, CD56-, CD16+, CD4-, CD8 dim+, CD7 dim+, CD5-
Detection of T Cell Neoplasia: Specific Populations
Absence of normal antigensPresence of abnormal antigens Abnormal antigen intensityAbnormal sized cellsIncreased Numbers of Specific
PopulationsRestricted Populations
Detection of Chronic Lymphoproliferative Disorders Without Specific IP
Restricted Populations are Abnormally Homogeneous
Clonal population- kappa/lambda or V Beta
Restricted B-Cell Populations: Kappa/ Lambda
SF12 945 pb_01_S-1.fcs SINGLETS
FSC-A
SSC
-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144
8c L FSC SSC
SF12 945 pb_08_B-6+ K-p.fcs SINGLETS
CD19 PC7
SSC
-A
102 103 104 105
0
65536
131072
196608
262144
8c B CD19 PC7 SSC
SF12 945 pb_05_B-2.fcs 8c L FSC SSC
CD19 PC7
CD5
Per
CP
Cy5
5
102 103 104 105
102
103
104
105 59.28% 0.03%
40.68% 0.00%
SF12 945 pb_05_B-2.fcs SINGLETS
CD19 PC7
CD5
Per
CP
Cy5
5
102 103 104 105
102
103
104
105
0.01%93.06%
0.01%6.92%
Kappa-p APC
Lam
bda-
p FI
TC
102 103 104 105
102
103
104
105
0.01% of leukocytes are CD19+. They are all kappa monoclonal and CD5+
CD5 PerCP Cy55
CD
38 P
E
102 103 104 105
102
103
104
105
CD43 APC
CD
81 F
ITC
102 103 104 105
102
103
104
105
CD20 AH7
CD
22 P
E
102 103 104 105
102
103
104
105
Restricted T Cell Populations: CD4/CD8
Coexpression of CD4 and CD8 Can be helpful but there are some normal double + T
CD 8
CD 4CD4
CD
8Restricted to CD4 +, CD8 –Not usually helpful in MRD
T-Cell Receptor
First DJ joining: joining of the Dβ1 gene segment to one of six Jβ1 segments or the joining of the Dβ2 gene segment to one of seven Jβ2 segments
Vβ-to-DβJβ rearrangement then occurs using one of the V Beta regions
There are a set number of beta V regions (V beta) that can be used
Generation of Beta Chain by V(D)J Joining
T Cell Vb Repertoire
Each T-cell has a single Vβ domaine used in its beta chain of the TCR. Clonal T-cells arise from a single T-cell and have the exact same Vβ whereas reactive T-cells have different ones.
There are Vβ-specific antibodies now that recognize 70% of all individual Vβ domains
We use an 8 tube panel 3 antibodies in 2 colors
CD3 PerCP
CD
8 A
PC
100
101
102
103
104
100
101
102
103
104
CD3+CD8+
Vb11
Vb14
Vb22
FITC
PECD3 PerCP
CD
4 A
PC
100 101 102 103 104100
101
102
103
104
CD3+CD4+
TCR Vβ Analysis
Utility of V Beta Analysis
FSC/SSC Lymphocyte Gate:
76 yo WF with chronic anemia- Hct 29.8, platelets 120K/MM3, WBC 2.09 with 78.7% lymphocytes
FSC-Height
SS
C-H
eigh
t
0 256 512 768 10240
256
512
768
1024
Gate 1
Consistent with T-Cell LGL: CD3+, CD16-, CD56-, CD57+, CD7 dim to-, CD2+, CD5+, CD8+Is it clonal?
CD3 APC
CD
56+1
6 P
E
100
101
102
103
10410
0
101
102
103
104
CD3 APC
CD
8 P
E
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
57 F
ITC
100 101 102 103 104100
101
102
103
104
CD5 APC
CD
2 P
E
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD
7 FI
TC
100
101
102
103
10410
0
101
102
103
104
V beta 9 FITC
V b
eta
16 P
E
100
101
102
103
104
100
101
102
103
104
0.43%95.42%
1.86%2.28%
V beta 17.1
V beta 18 FITC
V b
eta
20 P
E
100
101
102
103
104
100
101
102
103
104
0.51% 2.48%
94.62% 2.39%
V beta 5.1
V beta 13.1 FITC
V b
eta
8 P
E
100 101 102 103 104100
101
102
103
104
3.85%92.95%
0.80%2.40%V beta 13.6
V beta 5.3 FITC
V b
eta
3 P
E
100 101 102 103 104100
101
102
103
1042.34%
V beta 7.1
2.34% 0.64%
73.01% 24.02%
V beta 5.2 FITC
V b
eta
12 P
E
100 101 102 103 104100
101
102
103
1040.39% 2.67%
95.68% 1.26%
V beta 2
V beta 23 FITC
V b
eta
21.3
PE
100 101 102 103 104100
101
102
103
104
1.94%96.30%
1.46%0.30%
V beta 1
V beta 11 FITC
V b
eta
14 P
E
100 101 102 103 104100
101
102
103
1040.50% 1.43%
96.02% 2.04%
V beta 22
V beta 13.2 FITC
V b
eta
7.2
PE
100 101 102 103 104100
101
102
103
104
2.20%94.66%
0.98%2.16%
V beta 4
The T-Cell LGL is clonal, expressing Vb 5.3
CD3 PerCP
CD
8 A
PC
100
101
102
103
104
100
101
102
103
104
CD3+CD8+
FCI Diagnosis of Minimal T Cell Neoplasia:
CD3 PerCP
CD25
PE
100 101 102 103 104100
101
102
103
104
CD3 APC
CD4
FITC
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD7
FIT
C
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
26 F
ITC
100 101 102 103 104100
101
102
103
104A. Before Treatment
CD3 PerCP
CD7
FITC
100 101 102 103 104100
101
102
103
104
CD3 APC
CD4
Per
CP
100 101 102 103 104100
101
102
103
104
CD3 PerCP
CD25
PE
100 101 102 103 104100
101
102
103
104
CD3 APC
CD26
FIT
C
100 101 102 103 104100
101
102
103
104B. After Treatment
FCI Diagnosis of Minimal T Cell Neoplasia: V Beta Analysis
CD3 APC
CD
56+1
6 PE
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
19 P
erC
P
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
4 FI
TC
100 101 102 103 104100
101
102
103
104
CD7 FITC
CD
3 Pe
rCP
100 101 102 103 104100
101
102
103
104
CD26 FITC
CD
4 Pe
rCP
100 101 102 103 104100
101
102
103
104
CD3 APC
CD
8 PE
100 101 102 103 104100
101
102
103
104
Patient with history of T cell neoplasm: CD3 dim, CD7-, CD4-, CD8-, CD26-, and V Beta 22, post Tx
FCI Diagnosis of Minimal T Cell Neoplasia: V Beta Analysis
Patient with history of T cell neoplasm: CD3 dim, CD7-, CD4-, CD8-, CD26-, and V Beta 22, post Tx
V beta 3 FITC
V be
ta 5
.3 P
E
100 101 102 103 104100
101
102
103
1040.00% 1.50%
98.20% 0.30%
Vb 5.3
Vb 3
Vb 7.1
CD3 PerCP
CD
4+8
APC
100 101 102 103 104100
101
102
103
104
Adjust gate or replace gate to select abnormal cells
V beta 14 FITC
V be
ta 1
1 PE
100 101 102 103 104100
101
102
103
104
Vb 22
Vb 14
Vb 11
0.00% 82.39%
17.01% 0.60%
SF14 1070 pb_01_S-1.fcs 8c MNC
CD3 PerCP
CD
19 P
E
102 103 104 105
102
103
104
105 1.14% 0.03%
36.34% 62.49%
SF14 1070 pb_04_B-1.fcs 8c MNC
CD20 AH7
CD
103
FITC
102 103 104 105
102
103
104
105
1.15%98.17%
0.15%0.54%
SF14 1070 pb_07_B-4.fcs 8c MNC
CD19 PC7
CD
10 A
PC
102 103 104 105
102
103
104
105
SF14 1070 pb_07_B-4.fcs 8c MNC
CD19 PC7
CD
11c
V450
102 103 104 105
102
103
104
105
SF14 1070 pb_07_B-4.fcs 8c MNC
Kappa-m FITC
Lam
bda-
m P
E
102 103 104 105
102
103
104
105
SF14 1070 pb_07_B-4.fcs CD19 CD11c HCL?
Kappa-m FITCLa
mbd
a-m
PE
102 103 104 105
102
103
104
105
FCI Diagnosis of Minimal B Cell Neoplasia:
Flow Cytometric Detection of Lymphoma MRD
